Chemical compounds of various types can inhibit the growth of or kill microbes or other life forms. Mankind has exploited this phenomenon as exemplified by such everyday items as over-the-counter disinfectants and pharmaceutical compounds such as antibiotics. The invention that is the subject of this work deals with modification to and elaboration upon a recently discovered class of antimicrobial compounds. Unlike the common antimicrobial compounds exemplified above, the compounds making up this class are proteins also known as peptides. Proteins are made up of individual building blocks called amino acids. The amino acids are linked together by chemical links called peptide bonds. One end of the string of amino acids that makes up a protein is called the N-terminal end or amino terminal end, and the other end is called the C-terminal or carboxyl terminal end.
A particular peptide can be "built" in two ways. One way is to chemically synthesize the peptide using exclusively human, i.e., non-genetic intervention. Under this methodology the particular amino acid building blocks are selected and connected in the appropriate order using chemical reactions. This chemical synthesis of proteins does not directly involve biological or genetic assistance. The second method employs such assistance by manipulating the genetics of a cell system such that the system makes the desired protein. This latter methodology is commonly referred to as genetic engineering.
The antimicrobial peptides that are the subject of this invention, as will be set forth more fully in detail below, have been modified as taught by this invention so that they are especially useful in agricultural settings. Using the discoveries and teachings of this invention, it has been established that antimicrobial peptides can be used to retard and/or kill plant pathogens that have proved to be a nuisance, or worse destructive, to plants having either agronomic value or horticultural value. Among the practical applications of this invention are the application of these antimicrobial peptides to plants using traditional methods such as sprays, or non-traditional methods such as by genetically modifying or engineering plant cells such as corn or potatoes to express these peptides. For example, genetic material that codes for one of the antimicrobial peptides of this invention could be inserted into corn (maize) that ordinarily does not have genes for these peptides thereby conferring a high degree of plant pathogen resistance to the genetically transformed corn plant. In this connection it is significant that these antimicrobial peptides ordinarily are not found in plant cells. In either event the benefits to society from this invention are anticipated to be quite significant because the antimicrobial compounds set forth herein could significantly reduce, or in some cases eliminate, the need for costly, petroleum-derived pesticide compounds.
The antimicrobial peptides to which we have been referring were first reported in 1987 when two groups of researchers, one headed by Dudley Williams and one headed by Michael Zasloff, successfully characterized and reported a number of peptides derived from natural peptides which are secreted by glands contained within the skin of the African Clawed Frog, Xenopus laevis. See, Giovannini, et al., "Biosynthesis and Degradation of Peptides Derived from Xenopus Laevis Prohormones" Biochem. J. 243, (1987), 113-120; and Zasloff, "Magainins, A Class of Anti-microbial Peptides From Xenopus Skin: Isolation, Characterization of Two Active Forms and Partial cDNA Sequence of a Precursor," Proc. Natl. Acad. Sci. USA 84, (1987), 5449-5453. Their research was prompted, at least in part, by the observation that this species of frog has remarkable recuperative power and the ability to remain free from infection during wound-healing with little or no post-operative care.
Amongst these peptides, two 23 residue compounds, popularly named magainins, have become the subject of increasing attention. These are Magainin 1 having an amino acid sequence of Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Gly-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Gl u-Ile-Met-Lys-Ser, and Magainin 2 having replacements of Lys for Gly at position 10 and Asn for Lys at position 22 in the above sequence. Both Magainin 1 and Magainin 2 have been investigated for potential pharmaceutic use because of their broad spectrum antimicrobial activity against human pathogens. This is particularly true of Magainin 2.
In addition, a number of magainin based derivatives having varying degrees of activity have been produced and investigated. See Juretic, et al., "Magainin 2 Amide and Analogues, Antimicrobial Activity, Membrane Depolarization and Susceptibility of Proteolysis, " Febs Lett. 249, (1989), 219-223; Chen, et al., "Synthetic Magainin Analogues With Improved Antimicrobial Activity," Febs Lett. 236, (1988), 462-466; Chen et al, U.S. patent application Ser. No. 280,363, filed Dec. 6, 1988; Cuervo, et al., "Synthesis and Antimicrobial Activity of Magainin Alanine Substitution Analogs," Proceedings of the Eleventh American Peptide Symposium; Peptides: Chemistry, Structure and Biology (J. E. Rivier, et al.), (1990), pp. 124-126, published by ESCOM-Leiden, Neth.; Cuervo, et al., "The Magainins: Sequence Factors Relevant to Increased Antimicrobial Activity and Decreased Hemolytic Activity," Peptide Research 1, (1988), 81-86; World Patent Application No. WO 88/06597; and Japanese Patent Application No. JP-1/299,299. These include the complete single residue omission analogue series of Magainin 1 and 2, select N-terminal omissions of Magainin 2, as well as the complete alanine (Ala) replacement analog series of Magainin 2, and Magainin 2 derivatives which may be useful as an antibiotic and/or an anti-cancer drug and which are substituted at the 5th and 12th positions.
These magainin derivatives raise more questions about the nature, the properties, and characteristics of the structure and activity of magainins and magainin derived peptides than they answer. For example, both Zasloff, et al. and Cuervo, et al. have reported that omission analogs of magainins have reduced activity against animal pathogens. See, Zasloff et al., "Antimicrobial Activity of Synthetic Magainin Peptides and Several Analogs," Proc. Natl. Acad. Sci. USA 85, (1988), 910-913; and Cuervo, et al., "The Magainins: Sequence Factors Relevant to Increased Antimicrobial Activity and Decreased Hemolytic Activity" supra. Both research groups also appear to agree that the N-terminal region (amino acids 1-14) is critical for the activity of the peptide with regard to animal pathogens. However, there is no agreement on the extent to which omissions in this region affect the antimicrobial activity of the resulting peptide. Zasloff's group has established that magainin omission derivatives having only a single omitted amino acid at the amino terminus do not show appreciable decrease in activity. According to Zasloff's research, only when the resulting peptide is 19 residues or shorter (consecutive omissions from the N-terminus) is the decrease in activity significant, and/or total. This is in stark contrast to the findings of Cuervo's group. Cuervo et al. found that single residue omissions in the N-terminal region totally defeated the activity of Magainin 1 and Magainin 2 amide (--NH.sub.2).
These two research groups have also produced conflicting information with regard to the relative influence of single amino acid deletions on the carboxyl terminus of Magainin 2. Zasloff and his colleagues have demonstrated that removal of the Ser residue at the carboxyl end of Magainin 2 essentially eliminated activity against human bacterial pathogens, while Cuervo et al. reported only limited reduction in the activity of this single omission derivative of Magainin 2 against some of the same human pathogens. See M. Zasloff, U.S. Pat. No. 4,810,777; and Cuervo et al., "The Magainins: Sequence Factors Relevant To Increased Antimicrobial Activity and Decreased Hemolytic Activity," supra. Surprisingly, the present inventors have discovered that single and double residue omissions in the C-terminal (not N-terminal) region of magainins and magainin derived peptides can have profound effects on activity, especially with regard to efficacy against plant pathogens as opposed to animal pathogens.
The examination of certain substitution derivatives of natural magainins only exacerbates these issues of critical positions. Specifically, the N-terminal region of magainins and/or magainin-derived peptides is supposedly critical for activity. See Cuervo et al., "The Magainins: Sequence Factors Relevant To Increased Antimicrobial Activity and Decreased Hemolytic Activity," supra. In addition, a substitution of Ala in position 19 of the amide form of Magainin 2 yielded a five-fold increase in potency when compared to unsubstituted Magainin 2--NH.sub.2. This substitution was superior to all other Ala substitutions in positions 1-14. See, Cuervo et al. "Synthesis And Antimicrobial Activity of Magainin Alanine Substitution Analogs". supra; see, also, Chen et al., "Synthetic Magainin Analogs With Improved Antimicrobial Activity," supra (reporting increased antimicrobial activity of a Magainin 2 having alanine substituted in the 8th, 13th, and 18th positions thereof). Thus, no clear guidance is extant as to the specific modifications which would render such peptides useful in protecting plants from plant pathogens.
Much of the magainin literature has concentrated on the postulated mechanism by which magainin peptides inhibit microbial activity and cause lysis in, for example, protozoa. These papers have also discussed the interrelationship of the alpha-helix structure, size and charge attributed to these peptides and their utility as antimicrobial agents. See, generally, Matsuzaki, et al., "Magainin 1-Induced Leakage of Entrapped Calcein Out Of Negatively-Charged Lipid Vesicles," Biochimica et Biophysica Acta 981 (1989), 130-134; Rana, et al., "Outer Membrane Structure in Smooth and Rough Strains of Salmonella Typhimurium and Their Susceptibility to the Antimicrobial Peptides, Magainins and Defensins, " Prog. Clin. Biol Res. 292, (1989), 77-85; Chen, et al., "Magainin Analogs: A Study of Activity as a Function of Alpha-Helix Modification," Proc. of Eleventh American Peptide Symposium, supra, at pp. 124-126; Westerhoff, et al., "Magainins and the Disruption of Membrane Linked Free-Energy Transduction," Proc. Natl. Acad. Sci. USA 86, (1989), 6597-6601; and U.S. Pat. No. 4,810,777. See, also, Cannon, " A Family of Wound Healers," Nature 328, (1987), 478; Williams et al., "Raman Spectroscopy of Synthetic Antimicrobial Frog Peptides Magainin 2a and PGLa", Biochemistry 29, (1990), 4490-4496; Rana et al. "Interactions between Salmonella Typhimurium Lipopolysaccharide and the Antimicrobial Peptide, Magainin 2 Amide", FEBS Lett. 261, (1990), 464-467; Berkowitz et al., "Magainins: A New Family of Membrane-Active Host Defense Peptides", Biochemical Pharmacology 39, (1990), 625-629; Duclohier, et al., "Antimicrobial Peptide Magainin 1 from Xenopus Skin Forms Anion-Permeable Channels in Planar Lipid Bilayers," Biophys. J. 56, (1989), 1017-1021. See, also, Urrutia et al., "Spontaneous Polymerization of the Antibiotic Peptide Magainin 2," FEBS Lett. 247, (1989), 7-21.
The published works regarding magainins and other classes of antibiotic or antimicrobial peptides (for example, cecropins, defensins, sarcotoxins, melittins, and the like) of which the inventors are aware have generally centered on human pharmaceutical-related health technologies. Exceptions, however, include two applications filed by Jaynes et al., (WO 89/04371 and WO 88/0976) which generally relate to plants which have been genetically enhanced for disease resistance. Jaynes et al. have speculated without supporting data that genetically transformed plants may be produced which contain an expressible heterologeous gene for an antimicrobial peptide. In this way, it is hoped that the plant has enhanced resistance to disease. According to Jaynes et al., however, peptides such as melittins, bombinins, and magainins having less than about 30 residues are not preferred for use in crop protection applications, presumably since the host plant cells may be adversely affected by their incorporation and/or presences.
The preferred peptides in accordance with Jaynes et al. have from about 30 to about 40 amino acids because they are more specific for bacteria and fungi. Jaynes et al. also state that peptides having more than about 40 amino acids may not be sufficiently antimicrobial when used alone to provide a broad spectrum of antimicrobial protection. The approach of Jaynes et al. for protecting plants from plant pathogens appears to center on finding specific, naturally occurring peptides having a level of activity and specificity close to that considered advantageous and then to modify that peptide to optimize its characteristics. See also, Jaynes et al., "Increasing Bacterial Disease Resistance In Plants Utilizing Antibacterial Genes from Insects," BioEssays 6, (1987), 263-270.
Others have published information relating to the incorporation of antimicrobial peptides into plants or, in fact, the use of antimicrobial peptides to protect plants from plant pathogens. See, EPO 0,299,828; P. Casteels et al., "Apidaecins: Antibacterial Peptides From Honeybees," The EMBO J. 8, (1989), 2387-2391; F. Ebrahim-Nesbat et al., "Cutivar-Related Differences in the Distribution of Cell-Wall-Bound Thionins in Compatible and Incompatible Interactions Between Barley and Powdery Mildew," Planta 79, (1989), 203-210. However, the published information lacks a discussion regarding the various problems and solutions associated with the incorporation and/or use of such peptides with plants. It is desirable that the antimicrobial peptides of this invention are not only useful in protecting a plant from plant pathogens, but that the peptides in general are at least partially protected against plant proteases and do not significantly harm the very plant cells they are intended to protect.
Proteins produced in nature often comprise a Met amino acid bonded to the amino or N-terminal end. This is not the case with naturally occurring magainins or other naturally occurring antimicrobial peptides. Part of the present invention addresses synthesis of antimicrobial peptides with an N-terminal Met residue. Having synthesized such peptides, this invention also sets forth how such peptides are useful to protect plants from plant pathogens.
Prior to this invention, no one has considered modifying magainin based peptides such that they are both active against plant pathogens and particularly suited for use with and/or for incorporation into plants. Specifically, no one has considered the effect of naturally occurring plant enzymatic activity on antimicrobial peptides, i.e., the effect of plant proteolytic activity on the antimicrobial peptides such as those set forth in this invention; the potential deleterious effect of antimicrobial peptides on the plant cells the peptides are supposed to protect; or how such detrimental interactions can be ameliorated. Similarly, no one has squarely faced the impact of modified magainins on plant cell toxicity, also known as phytotoxity.
The present invention therefore addresses not only the need for antimicrobial peptides active against at least one plant pathogen, but also addresses the need for peptides which are specifically designed to operate in the plant kingdom. As a result of this invention, it has been determined that Magainin 1 derivatives are generally lower in phytotoxicity. Consequently, this invention shows that these derivatives are especially useful in agricultural and agronomic settings in which the peptides could be used on the plants, e.g., as a spray, or could be incorporated into a plant, e.g., genetically engineering a plant cell so that the plant cell itself produces the peptide. Further, it has been unexpectedly found that certain bonds between the amino acid constituents of magainins are sensitive to proteolytic degradation by at least one plant protease. The present inventors have also developed antimicrobial peptides which are resistant to such degradation, and have shown that changes to effect such resistance do not compromise antimicrobial activity and do not increase phytotoxicity.